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Team:Newcastle/9 July 2010
From 2010.igem.org
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===Transformation=== | ===Transformation=== | ||
| + | Aim: to transform competent ''E. coli'' DH5alpha with pSB1AT3 vectors containing the lacI insert (in the ratios of 1:3 and 1:5 of vector to insert) | ||
Link to transformation protocol (to be added) | Link to transformation protocol (to be added) | ||
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# Add 1 ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time). | # Add 1 ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time). | ||
# Plate out 200 µl/plate on LB (agar at 1.5%). | # Plate out 200 µl/plate on LB (agar at 1.5%). | ||
| - | #* | + | #* |
# Incubate plates overnight at 37°C. | # Incubate plates overnight at 37°C. | ||
Revision as of 14:46, 21 July 2010
Transformation
Aim: to transform competent E. coli DH5alpha with pSB1AT3 vectors containing the lacI insert (in the ratios of 1:3 and 1:5 of vector to insert) Link to transformation protocol (to be added)
Tubes used:
Each of the following five tubes contain 200 µl of competent E. coli DH5alpha. To this the DNA to be transformed was added.
- 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
- 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
- Negative control for ligation (contains vector with no insert)
- Control for transformation (without plasmid)
- Control for transformation (with plasmid, pSB1AT3)
Protocol:
- Thaw a 200 µl aliquot of E. coli DH5alpha and add the transforming DNA. For tubes 1-3 5 µl of vector was added, for tube 4 no vector was added and for tube 5 only 1 µl of vector was added. This is because tubes 1 to 3 have been ligated and will contain cells in different forms of the ligated vector whereas tube 5 contains the ligated vector (our control).
- Incubate for 45 mins on ice.
- Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
- Add 1 ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
- Plate out 200 µl/plate on LB (agar at 1.5%).
- Incubate plates overnight at 37°C.
